The present invention relates to the cell surface receptors for heat shock proteins (HSPs), such as gp96, Hsp70 and Hsp90, cells that express the Hsp receptor, genes that encode the Hsp receptor, and antibodies and other molecules that bind the receptor. The invention also relates to the diagnostic uses of these molecules in immunotherapy. HSP cell surface receptors recognize and bind to HSPs and are associated with the cell membranes of a subset of macrophages and dendritic cells. HSP cell surface receptors can have uses in the diagnosis and treatment of cancer and proliferative diseases.
Heat shock proteins (HSPs), also referred to as stress proteins, were first identified as proteins synthesized by cells in response to heat shock. To date, five families of HSP have been identified based on molecular weight, Hsp 100, Hsp90, Hsp70, Hsp60, and smHsp. Many members of these families were found subsequently to be induced in response to other stressful stimuli including nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular pathogens. (See Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science 260:1902-1903; Gething et al., 1992, Nature 355:33-45; and Lindquist et al., 1988, Annu. Rev. Genetics 22:631-677).
The major HSPs can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have not been stressed. For example, the highly inducible mammalian Hsp70 is hardly detectable at normal temperatures but becomes one of the most actively synthesized proteins in the cell upon heat shock (Welch et al., 1985, J. Cell. Biol. 101:1198-1211). In contrast, Hsp90 and Hsp60 proteins are abundant at normal temperatures in most, but not all, mammalian cells and are further induced by heat (Lai et al., 1984, Mol. Cell. Biol. 4:2802-2810; van Bergen en Henegouwen et al., 1987, Genes Dev. 1:525-531).
Studies on the cellular response to heat shock and other physiological stresses revealed that the HSPs are involved not only in cellular protection against these adverse conditions, but also in essential biochemical and immunological processes in unstressed cells. HSPs accomplish different kinds of chaperoning functions. For example, members of the Hsp70 family, located in the cell cytoplasm, nucleus, mitochondria, or endoplasmic reticulum (Lindquist, S. et al., 1988, Ann. Rev. Genetics 22:631-677), are involved in the presentation of antigens to the cells of the immune system, and are also involved in the transfer, folding and assembly of proteins in normal cells. HSPs are capable of binding proteins or peptides, and releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH.
Other stress proteins involved in folding and assembly of proteins include, for example, protein disulfide isomerase (PDI), which catalyzes disulfide bond formation, isomerization, or reduction in the endoplasmic reticulum (Gething et al., 1992, Nature 355:33-45).
Heat shock proteins are among the most highly conserved proteins in existence. For example, DnaK, the Hsp70 from E. coli has about 50% amino acid sequence identity with Hsp70 proteins from excoriates (Bardwell et al., 1984, Proc. Natl. Acad. Sci., 81:848-852). The Hsp60 and Hsp90 families also show similarly high levels of intra-family conservation (Hickey et al., 1989, Mol. Cell. Biol., 9:2615-2626; Jindal, 1989, Mol. Cell. Biol., 9:2279-2283). In addition, it has been discovered that the Hsp60, Hsp70 and Hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress.
Srivastava et al. demonstrated immune response to methylcholanthrene-induced sarcomas of inbred mice (1988, Immunol. Today 9:78-83). In these studies, it was found that the molecules responsible for the individually distinct immunogenicity of these tumors were glycoproteins of 96 kDa (gp96) and intracellular proteins of 84 to 86 kDa (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich et al., 1986, Proc. Natl. Acad. Sci. USA 83:3121-3125). Immunization of mice with gp96 or p84/86 isolated from a particular tumor rendered the mice immune to that particular tumor, but not to antigenically distinct tumors. Isolation and characterization of genes encoding gp96 and p84/86 revealed significant homology between them, and showed that gp96 and p84/86 were, respectively, the endoplasmic reticular and cytosolic counterparts of the same heat shock proteins (Srivastava et al., 1988, Immunogenetics 28:205-207; Srivastava et al., 1991, Curr. Top. Microbiol. Immunol. 167:109-123). Further, Hsp70 was shown to elicit immunity to the tumor from which it was isolated but not to antigenically distinct tumors. However, Hsp70 depleted of peptides was found to lose its immunogenic activity (Udono and Srivastava, 1993, J. Exp. Med. 178:1391-1396). These observations suggested that the heat shock proteins are not immunogenic per se, but form noncovalent complexes with antigenic peptides, and the complexes can elicit specific immunity to the antigenic peptides (Srivastava, 1993, Adv. Cancer Res. 62:153-177; Udono, H. et al., 1994, J. Immunol., 152:5398-5403; Suto et al., 1995, Science, 269:1585-1588).
Noncovalent complexes of HSPs and peptide, purified from cancer cells, can be used for the treatment and prevention of cancer and have been described in PCT publications WO 96/10411, dated Apr. 11, 1996, and WO 97/10001, dated Mar. 20, 1997 (see also copending U.S. patent applications Ser. No. 08/796,318 (now U.S. Pat. No. 6,017,540) filed Feb. 7, 1997 by Srivastava and Chandawarkar and Ser. No. 08/796,316 (now U.S. Pat. No. 5,830,464) filed Feb. 7, 1997 by Srivastava, each of which is incorporated by reference herein in its entirety). Stress protein-peptide complexes can also be isolated from pathogen-infected cells and used for the treatment and prevention of infection caused by the pathogen, such as viruses, and other intracellular pathogens, including bacteria, protozoa, fungi and parasites. See PCT publication WO 95/24923, dated Sep. 21, 1995. Immunogenic stress protein-peptide complexes can also be prepared by in vitro complexing of 30 stress protein and antigenic peptides, and the uses of such complexes for the treatment and prevention of cancer and infectious diseases has been described in PCT publication WO 97/10000, dated Mar. 20, 1997. The use of stress protein-peptide complexes for sensitizing antigen presenting cells in vitro for use in adoptive immunotherapy is described in PCT publication WO 97/10002, dated Mar. 20, 1997.
Stress protein-peptide complexes have been purified as described previously; see for example, PCT Publication WO 95/24923, dated Sep. 21, 1995. For the purpose of preparing a vaccine against cancer, the amount of immunogenic material obtainable for use is directly related to the amount of starting cancer cells. Since only a small number of cancer cells can be obtained from a subject, especially if the cancer is at an early stage, the supply of cancer cells for producing the HSP-peptide complex is often very limited. Because of this limited supply of cancer cells, the development of new techniques are needed to aid in the process of purifying recombinant HSP-peptide complexes for use in immunotherapy.
For commercial production of a vaccine or therapeutic agent, a constant supply of large amounts of HSP-peptide complexes is advantageous. Thus, there is a need for a dependable long-term source of HSP-peptide complexes that does not depend on availability of fresh cell samples from cancer patients. Readily available purified components of the molecular machinery involved in the elicitation of specific immunity by heat shock protein-peptide complexes will greatly enhance immunotherapeutic techniques when only a very small amounts of tumor tissue is available from a patient.
Major histocompatibility complex (MHC) molecules present antigens on the cell surface of antigen-presenting cells. Cytotoxic T lymphocytes (CTLs) then recognize MHC molecules and their associated peptides and kill the target cell. Antigens are processed by two distinct antigen processing routes depending upon whether their origin is intracellular or extracellular. Intracellular or endogenous protein antigens, i.e., antigens synthesized within the antigen-presenting cell, are presented by MHC class I (MHCI) molecules to CD8+ cytotoxic T lymphocytes. On the other hand, extracellular or exogenously synthesized antigenic determinants are presented on the cell surface of xe2x80x9cspecializedxe2x80x9d or xe2x80x9cprofessionalxe2x80x9d APCs (macrophages, for example) by MHC class II molecules to CD4+ T cells (see, generally, Fundamental Immunology, W. E. Paul (ed.), New York: Raven Press, 1984). This compartmental segregation of antigen processing routes is important to prevent tissue destruction that could otherwise occur during an immune response as a result of shedding of neighboring cell MHCI antigens.
The capacity of gp96-peptide complexes to elicit an immune response is dependent upon the transfer of the peptide to MHC class I molecules of antigen-presenting cells (Suto and Srivastava, 1995, supra). Endogenously synthesized antigens chaperoned by gp96 in the endoplasmic reticulum [ER] can prime antigen-specific CD8+ T cells (or MHC I-restricted CTLs) in vivo; this priming of CD8+ T cells requires macrophages. However, the process whereby exogenously introduced gp96-peptide complexes elicit the antigen-specific CD8+ T cell response is not completely understood since there is no established pathway for the translocation of extracellular antigens into the class I presentation machinery. Yet antigenic peptides of extracellular origin associated with HSPs are somehow salvaged by macrophages, channeled into the endogenous pathway, and presented by MHC I molecules to be recognized by CD8+ lymphocytes (Blachere et al., 1997, J. Exp. Med., 186:1315-22).
Little is known about the route the peptides take inside the cell before reaching the class I molecules. There currently exists several proposed mechanisms for the delivery of extracellular peptides to the MHC I molecules for presentation. One model proposed to explain this apparent paradox is that HSP-chaperoned peptides, or fragments thereof, are transferred to MHC I molecules on the cell surface of macrophages, which internalize them and re-present these antigenic peptides to CD8+ T lymphocytes. Another model suggest that soluble extracellular proteins can be trafficked to the cytosol via constitutive macropinocytosis in bone marrow-derived macrophages and dendritic cells (Norbury et al., 1997, Eur. J. Immunol. 27:280-288). Yet another hypothetical model attempts to explain the phenomenon by suggesting that HSPs are taken up by the MHC class I molecules of the macrophage, which finally stimulate the appropriate T cells (Srivastava et al., 1994, Immunogenetics 39:93-98. There is also the hypothesis that the mannose receptor is used in the uptake of gp96 but no mechanism has been proposed for the non-glycosylated HSPs, such as HSP70 (Ciupitu et al., 1998, J. Exp. Med., 187:685-691). Others suggested that a novel intracellular trafficking pathway may be involved for the transport of peptides from the extracellular medium into the lumen of ER (Day et al., 1997, Proc. Natl. Acad. Sci. 94:8064-8069; Nicchitta, 1998, Curr. Opin. in Immunol. 10:103-109). Further suggestions include the involvemnt of phagocytes which (a) possess an ill-defined pathway to shunt protein from the phagosome into the cytosol where it would enter the normal class I pathway; (b) digest ingested material in lysosomes and regurgitate peptides for loading on the surface to class I molecules (Bevan 1995, J. Exp. Med. 182:639-41).
A better understanding of this process and the characterization of specific molecules involved in the uptake of HSPs or HSP-peptide complexes could provide useful reagents and techniques for eliciting specific immunity by HSP and HSP-peptide complexes. The isolation of the heat shock protein receptor and the gene encoding thereof is instrumental to our understanding of the antigen presentation process and the development of novel diagnostic and therapeutic methods.
The present invention is based on the discovery of a receptor that recognizes and binds to heat shock protein. The existence of such an HSP receptor on the cell surface was unexpected because HSPs/stress proteins are generally known to be cytoplasmic and are also known to be very abundant. The receptor is associated with the cell membranes of a subset of macrophages, dendritic cells, and possibly other cell types.
In one embodiment, the invention provides methods for enriching and isolating cells that express the HSP receptor.
In another embodiment, the invention provides methods for isolating the HSP receptor protein. The HSP receptor can be isolated from extracts of HSPR positive cells, and preferably fractions containing the membrane components of HSPR positive cells. Detection of the HSP receptor is accomplished by using antibodies that bind the HSP receptor, or by the assaying for HSP-binding activity. Isolated HSPR protein and fragments thereof are also encompassed by the invention. The invention also provides for antibodies to HSPR positive cells and HSPR protein, and fragments thereof.
In yet another embodiment, the invention provides methods for identifying and isolating nucleic acid molecules encoding HSP receptor, and fragments thereof. Methods to identify such nucleic acid molecules in HSPR positive cells include subtractive hybridization methods, DNA chip technologies, and differential display. To facilitate isolation of the HSPR cDNA, RNA from HSPR positive cells can be used to prepare a cDNA library. Such gene libraries can be screened by hybridization using oligonucleotide probes encoding a fragment of HSPR, or nucleic acid molecules encoding a homologous HSPR. Alternatively, functional screening or expression cloning methods can be applied to screen the libraries. The libraries are constructed and introduced into host cells such that the proteins encoded by the cDNAs are expressed. Labeled antibodies to HSPR or labeled HSP can be used to isolate clones in such gene expression libraries that express a functional HSPR, or a functional portion thereof. hspr gene, and fragments thereof, isolated by the methods of the invention are also encompassed.
The present invention further provides methods of use of the HSPR positive cells, HSPR protein, HSPR antibodies, and hspr gene. HSP receptors may serve to recognize and transport HSP-antigenic peptide complexes for the purpose of presenting such antigenic molecules to cells of the immune system and eliciting an immune response. Thus, HSPR may be used for modulating the immune response. Methods for identifying a molecule that enhances or blocks the function of HSPR are included in the invention. The compositions of the invention may be used in various diagnostic and therapeutic applications in the area of cancer and infectious diseases.